1. Technical Field of the Invention
This invention relates to the development of an efficient, high yield method for the physical purification of specific biological material. This invention relates particularly to a method for separating and isolating biological materials by labelling the biological materials with a binding composition. The material to be labelled can be reversibly anchored to a support before labeling. A binding composition preferably comprises an indicator such as a luminescent material or a magnetic particle. Contemplated indicators are appropriate for use in separating and isolating biological material in the microscopic range, e.g. chromosomes, mitochondria, chloroplasts, Golgi apparatus and other cellular organelles. A binding composition is preferably a nucleic acid probe or an antibody. After release from the support, the labelled material is isolated by sorting by means of the indicator.
2. Background of the Invention
Cells are composed of heterogeneous components. Individual components need to be separated prior to many investigations and procedures. Separation methods vary depending on the absolute and relative sizes of the material to be separated, and the degree of purity which must be achieved. To separate relatively large biological materials, that is, the "meat" of the stew, centrifugation, filtering and density gradient sedimentation are some of the relatively crude methods that are appropriate. To separate small, minute cellular components, the "spices" (e.g., chromosomes) flow cytometry has been used. Some materials, e.g., the Golgi apparatus, have not been satisfactorily separated by any means as an individual, intact cellular component.
Major efforts are currently being made to map and sequence the genomes of man, mouse, and several other selected organisms. Although genetic mapping methods can tell us a great deal about the organization of a genome, it is also necessary to isolate chromosome-specific DNA to obtain contiguous DNA sequence information and construct a physical map. This is usually done by cloning or screening cosmid, YAC, or the recently developed P1 libraries (Burke et al., 1991; Sternberg, 1992). However, the usefulness of all libraries is subject to specific limitations of insert size and cloning bias, i.e., the mouse genome contains DNA sequences that are apparently resistant to cloning in either E. coli or yeast (Little, 1990; Gibson et al., 1987). It is also now known that high levels of rearrangement and deletion occur frequently within YAC clones (Little, 1990).
A physical purification method would permit the isolation and sequencing of DNA that is otherwise unstable in conventional hosts. In addition, effective chromosome purification combined with chromosome breakage methods such as microdissection (Weber et al., 1990) or the use of artificial restriction enzymes (Moser et al., 1987), would in theory allow the construction of libraries for specific chromosomal subregions such as centromeres, repeated genes, translocation breakpoints, or the rearrangements involved in neoplastic progression. Applications of the present invention are not limited to the types of experiment outlined above, however. Even where straightforward library construction is the goal, physical purification methods will permit the rapid construction of highly enriched libraries, increasing the efficiency with which desired sequences can be cloned. This technology is applicable to many species besides the mouse and would be of great utility in the analysis of the human genome.
Various organelles and intracellular structures have proven difficult or impossible to isolate or purify by traditional cell fractionation methods such as density sedimentation. In addition, existing procedures for routine organelle isolations may disrupt or impair their physiology, and in some cases provide populations of organelles which are not developmentally representative (Dahlin and Cline, 1991). Antibodies specific to exposed epitopes on many intracellular compartments and structures have been reported (e.g. Ohba and Schatz, 1987; Pain et al., 1988; Yoneda et al., 1988;) and their specificity has been demonstrated immunocytochemically.
Most currently applied cell fractionation procedures exploit obvious differences in physical parameters between organelles and rely on centrifugation which generates large, damaging shear forces. Consequently, it is difficult to prepare physiologically intact subcellular fractions where only minor differences exist in physical properties or when the compartments are especially fragile to shear. Furthermore, most existing procedures for routine organelle isolations are lengthy, rendering some physiological observations difficult. The study of plastids and their metabolism has focused on mature green chloroplasts with considerably less attention given to the chloroplast developmental sequence from proplastids and even less to research on nongreen plastids (e.g. chromoplasts, amyloplasts, etc.). In many cases the nongreen plastids have a distinct metabolism which provides a superior physiological system for the study of many plant processes, such as carotenoid biosynthesis in chromoplasts, or starch synthesis in amyloplasts. Although intact mature green chloroplasts can be readily isolated by density sedimentation, the study of nongreen plastids has been encumbered by their fragile properties and difficulties in isolation from the tissues in which they develop.
Immunoabsorption of subcellular components by organelle specific antibodies bound to an appropriate [solid phase] support obviates some of these difficulties (Howell et al., 1988) and provides the possibility for separation from whole cell lysates by specific biochemical criteria. Various preparations of magnetic particles with distinct magnetic properties, surface characteristics, and mean diameter size ranges have been produced (Haukanes and Kvam, 1993.) These differences significantly influence their specific utility for biological separations, and even though magnetic cell purifications were achieved several years ago, magnetic particles with the necessary qualities needed for other types of separations were developed only very recently (Haukanes and Kvam, 1993).
Superparamagnetic (magnetic only in a magnetic field) microparticles (sized in the micron range) are synthesized by polymerizing polystyrene or polyacrolein in the presence of a magnetite ferrofluid (Ugelstad and Berge, 1988) or by formation of an agglomerate by silanation of a ferrofluid (Whitehead et al., 1985). With both of these preparations antibodies can be covalently coupled by the resultant surface character of the particles. Magnetic microparticles have been used in cell separation, immunoassays, isolation, identification and genetic analysis of specific nucleic acid sequences, and for isolation of DNA binding proteins (Haukanes and Kvam, 1993). The present inventors have examined the possibility of using similar magnetic microparticles as a solid support for immunoabsorption of intact plastids from whole cell lysates because their relatively high magnetic moment allows ease of separation using simple rare earth magnets.
Another class of particle preparations can be described as magnetic nanoparticles (ferrofluid derivatives sized in the nanometer range) consisting of ferric oxide crystals encapsulated by dextran (Molday and MacKenzie, 1982), which are activated with cyanogen bromide and subsequently coupled to protein preparations with diaminohexane. Magnetic nanoparticles have been shown to have a much faster binding reactivity than magnetic microparticles for intact mammalian cell separations (Miltenyi et al., 1990; Liberti and Feeley, 1991), hence greater specificity, and they do not aggregate in a magnetic field. The small size of nanoparticle preparations renders them applicable for subcellular immunolocalizations by allowing the antibodies carried by the particles to react with all exposed epitopes over the entire surface of the organelle. The immunoreactions occur as rapidly as if free in solution with a minimal degree of stearic hindrance compared with larger solid phase surfaces which can only react with a tangential surface. However, magnetic nanoparticles have an extremely low magnetic moment since the subparticles of ferromagnetic material are smaller than the domain size required for rapid magnetic precipitation and therefore, migrate only very slowly in conventional ferromagnetic fields. Consequently, organelles labelled with magnetic nanoparticles must be separated in a magnetic affinity column (Miltenyi et al., 1990). Disclosed herein is the use of antibodies specific to exposed epitopes of proteins on chloroplast outer envelopes coupled to magnetic nanoparticles to immuno-isolate various plastid subtypes from whole cell lysates.
Although there are many variations on the flow cytometry theme, the basic principle of this method is to label the cellular material, for example, chromosomes, according to its DNA content, which will be generally correlated with size, and to separate the material into collecting tubes by laser beams that quantitatively measure the DNA content. Flow separation of human chromosomes has been somewhat successful in completely separating some of the 23 pairs of chromosomes but has resulted in some aggregation of chromosomes of similar sizes, e.g., the human chromosomes 21-22 and Y. Therefore, this method is of limited usefulness.
Flow cytometry techniques are used for both analysis and sorting of biological macromolecules and cells (Darzynkiewicz & Crissman, 1990). Flow cytometry currently is the primary method for the purification of specific chromosomes. However, this method suffers from several drawbacks, and its efficacy is limited by the amount of time (hours or even days) required to sort large quantities of a single chromosome. For example, it is not yet possible to reliably separate different chromosomes whose DNA contents are similar and there is some cross-contamination of chromosomes having similar sizes.
Furthermore, flow cytometry techniques cannot be employed to separate individual chromosomes by DNA content alone in species such as the mouse whose karyotype consists of similarly sized telocentric chromosomes (these are chromosomes with their primary constrictions, the centromeres, located at one end). In such cases, somatic cell hybridization may be employed, although this is very laborious. In somatic cell hybridization, chromosomes may be isolated in a genetically different background by cell fusion and selection.
Although some attempts at developing physical isolation methods have been made (Dudin et al., 1988; Kandpal et al., 1990), there is no effective method for obtaining purified mouse chromosomes. Flow cytometry is currently the only available method for physically isolating specific whole chromosomes (Carrano et al., 1979). Maps constructed by analyzing cosmid libraries derived from flow-sorted material are currently being used for the analysis of the human genome (Van Dilla et al., 1989). Flow sorting is not generally feasible for mouse chromosomes, however, due to their uniform DNA content. Somatic cell hybrids containing a given chromosome, or cell types with nonstandard karyotypes, such as are found in mouse strains carrying single Robertsonian translocation chromosomes, have been used to differentiate the DNA content of mouse chromosomes. Furthermore, the efficacy of flow cytometry is limited by the time required to sort large quantities of a given chromosome. Finally, debris and cross contamination in sorted preparations limit their utility. Thus an alternative to FACS must be available before easy purification of mouse chromosomes can become a reality.
The availability of chromosome-specific, repetitive sequence hybridization probes (Moyzis et al., 1987) presents the possibility of a unique alternative method for chromosome isolation. The combination of in-situ hybridization of such probes, appropriately labelled (Narayanswami et al., 1992), to chromosome mixtures, followed by isolation based on the presence of the hybridized probe, should permit isolation of a specific chromosome without flow sorting. There has been one attempt to develop such a technique (Dudin et al., 1988). In that report, in-situ hybridization was carried out on suspensions of chromosomes prepared as for flow cytometry. Hybrids were labelled with large magnetic beads several microns in diameter. However, that approach was not very successful due to problems with adventitiously adsorbed contaminants, chromosome aggregation, and losses during centrifugation steps.